Direct cell-to-cell communication is mediated by channels (at gap junctions) that extend across two plasma membranes and a narrow extracellular space (gap) and result from the extracellular interactions between hemichannels (connexons). Each connexon is an aggregate of six identical proteins (connexins). Recently, much has been learned on gap junction architecture, permeants' nature, connexin chemical structure, channel electrical properties, etc. Yet, channel permeability regulation and connexin-connexin interaction are poorly understood. Channel closure (cell uncoupling is primarily a safety mechanism for cell survival, but it can also be a pathological process that disrupts cell cooperation (cardiac arrhythmias, dystocia, etc.). The long range goal of this proposal is to improve our understanding of the mechanism controlling channel permeability, and of the nature of extracellular connexin-connexin interactions. To this aim, we have designed a multidisciplinary approach that includes site-directed mutagenesis of connexins, expression of normal and mutant channels in oocyte pairs and in a gap junction incompetent cell line (SKHep1), measurement of junctional conductance (g/j) in oocytes by double voltage clamp (DVC), and of both g/j and single-channel conductance gamma(j) in SKHep1 and Novikoff hepatoma cells by double whole-cell clamp (DWCC, and channel reconstitution into liposomes and planar lipid bilayers with connexins isolated from rat heart and liver. For studying channel regulation, some projects will focus on the mechanism of Ca2+-induced uncoupling and the possible involvement of calmodulin (CaM) or a CaM-like protein, either directly or via kinases of phosphatases. These will include expression in oocytes and SKHep1 cells of connexins genetically modified at a potential CaM-binding site that we have recently identified, and testing of CaM mutants and CaM binding peptides in Novikoff hepatoma cells and reconstituted systems. Others projects will probe the uncoupling mechanism of general anesthetics by testing their potential interaction with amphiphilic connexin domains and the effects of xanthines and aminopyridines. The nature of extracellular connexin-connexin interactions will be studied by expressing connexins genetically modified at potential sites for Ca2+- bridges and by monitoring the formation of cell-cell channels between Novikoff cells. The hypothesis suggesting a mostly hydrophobic connexin domain as part of the external "channel seal" will be tested by expressing appropriate connexins mutants.